Veterinary Andrology and Reproductive Techniques

PRE REQUISITES

(Materials required, Preparation of stock Giemsa stain, Preparation of working Giemsa stain, Preparation of Sorenson's buffer and Preparation of 5% formaldehyde)

Giemsa stain is used to stain the sperms for assessing acrosome integrity.

Materials required

• Stock Giemsa stain
• Semen (fresh/frozen)
• Glass slides
• 5% formaldehyde

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Preparation of stock Giemsa Stain

Chemical	Quantity
Giemsa powder	1 gm
Glycerl	60 ml
Methanol	66 ml

• Weigh the Giemsa powder, put in a pestle and mortar.
• The glycerol is added slowly and grind it well. Then add methanol slowly and grind it.
• Finally the solution is filtered and stored in an amber colored bottle.

Stock Giemsa stain can be prepared and stored for years.

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Preparation of working Giemsa solution

• It is prepared immediately prior to use

Chemical	Quantity
Stock Giemsa stain	3 ml
Sorenson’s buffer	2 ml
Distilled water	45 ml

Woking Giemsa stain is prepared on the day of staining immediately before use. It cannot be stored like stock Giemsa solution

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Preparation of Sorenson’s Buffer

Chemical	Quantity
Na2HPo4.2H2O (21.682 gm/500 ml)	200 ml
KHPo4H2O (22.254 gm/500 ml)	80 ml
Stock buffer	280 ml

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Preparation of 5% formaldehyde

Chemical	Qantity
Formalin (37-41% W/V)	12.5 ml
Distilled water	87.5 ml